Toll-like receptors (TLRs) are key pathogen-recognition receptors in human innate immune cells that detect diverse pathogens from microbes and thus protect the body against infection. In human cells, ten TLRs (TLR1 to TLR10) have been identified, each with their own specificities. TLR3 recognizes viral double-stranded (ds) RNA derived from the genomes of dsRNA viruses, or generated during the replication of many viruses. Activation of TLR3 induces a strong Th1 immune response, including increased secretion of inflammatory cytokines and interferons, and subsequently leads to the maturation, differentiation, and proliferation of immune cells. Because these potent immune adjuvant effects facilitate eradication of viruses and malignant cells in the body, there has been intense interest in developing TLR3 activators for therapeutic applications. Synthetic double-stranded RNAs (dsRNAs) such as polyinosinic-polycytidylic (Poly (I: C) mimics viral dsRNA in activation of TLR3. These synthetic dsRNAs have been shown to increase resistance to viral infection and inhibit tumor growth in animal models, and their therapeutic effects are being evaluated in clinical trials. However, despite their beneficial effects, further development of these dsRNAs into more effective therapeutic agents is restricted firstly by their size;dsRNAs have a molecular size greater than 8,000 Da. Secondly, because of their size, it is difficult to further modify these dsRNAs into an oral pro-drug format. Thirdly, these dsRNAs lack specificity and are recognized by multiple cellular targets including RIG-I-like receptors and protein kinase PKR, in addition to the TLR3. This could explain, at least in part, the adverse effects of these dsRNAs. Finally, to date no small molecular weight activator(s) has been identified for TLR3, which has made it difficult to elucidate the structure-functional relationship between TLR 3 and its activators. For these reasons, discovery of novel small molecular weight TLR3 activators is necessary not only for the development of safer and more effective TLR3-based therapeutic agents but also for the development of a more specific activator of TLR3 to probe its biological function. Targeting TLR3 to screen chemical compound libraries is one of the best approaches to identify lead compounds for development of novel TLR3 activators. In this project, we plan to develop a stable cell line based TLR3 activation assay, and validate the robustness, reproducibility, and sensitivity of this assay in a 384-well plate format in order for this assay to be adapted into the automated processing procedures of a high throughput screening. The developed assay will be submitted to the NIH Molecular Libraries Production Center Centers Network (MLPCN) for implementation. We believe that this proposed work is a first step to discover novel lead compounds for TLR3 activation, which will lead to the development of highly specific activators to probe the biological function of TLR3, as well as the development of a new generation of TLR3-based therapeutic agents for treatment of cancers and infectious diseases. PUBLIC HEALTH RELEVANCE: Toll-like receptor 3 (TLR3) recognizes viral double-stranded (ds) RNA. Activation of TLR3 induces a strong Th1 immune response. Because this potent immune adjuvant effect facilitates eradication of viruses and malignant cells in body, there has been intense interest in developing TLR3 activators for therapeutic applications. In this project, we plan to develop a cell-based assay for high throughput screening to discover novel lead compounds for TLR3 activation, which will lead to the development of highly specific activators to probe the biological function of TLR3, as well as the development of a new generation of TLR3-based therapeutic agents for treatment of cancers and infectious diseases.